1. Field of the Invention
The present invention relates to a recombinant DNA prepared by linking a gene encoding an antigenic determinant of Escherichia coli (E. coli) to a CTXA2B gene of Vibrio cholerae or a LTXA2B gene of E. coli, and an expression plasmid including the recombinant DNA. Also, the present invention is concerned with a microorganism transformed with the expression vector. Further, the present invention relates to a vaccine protein against E. coli responsible for urinary tract infections, which is produced by the transformant.
In detail, the present invention relates to a novel vaccine against E. coli responsible for urinary tract infections (uropathogenic E. coli). The vaccine is a recombinant fusion protein which is prepared by linking by genetic recombination a gene encoding an antigenic determinant of uropathogenic E. coli to a CTXA2B gene encoding nontoxic A2 and B subunits of cholera toxin of Vibrio cholerae or a LTXA2B gene encoding nontoxic A2 and B subunits of E. coli heat-labile enterotoxin, wherein a translation product of the CTXA2B or LTXA2B gene serves as an adjuvant stimulating mucosal immune responses, expressing the resulting recombinant gene in E. coli, and isolating and purifying an expressed recombinant chimeric protein.
2. Description of the Prior Art
Urinary tract infections, which are bacterial infections common in the urinary tract, etc., present clinically as cystitis, pyelititis, acute chronic pyelonephritis, and the like (Kunin, 1994; Haley et al., 1985). Diseases caused by bacterial infections of the urinary tract do not exhibit clinical symptoms until a large number of microorganisms proliferate in the urinary tract, and their development frequency follows the most common respiratory tract diseases. Urinary tract infections are reported to be caused by invasion of ascending bacteria through the lower urinary tracts (Patton et al., 1991). Hematogenous infections may occur by Staphylococcus aureus, fungi, Mycobacterium tuberculosis, etc. Urinary tract infections are caused by various factors including urinary tract obstruction caused by pregnancy, calculus, etc., neurogenic bladder, vesicoureteral reflux, renal diseases, hypertension, diabetes mellitus, catheter insertion, and administration of analgesics and antipyretics (Kunin, 1994). On the other hand, cystitis and nephritis are common in women, while cystitis also occurs frequently in children, and urethritis is common in men (Haley et al., 1985). The urinary tract infections may accompany complications, such as renal papillary necrosis, renal abscess and perirenal abscess (Stamm et al., 1993). About 70% or higher of these urinary tract infections have been known to be caused by E. coli. According to some reports, due to these diseases, over seven million people visit primary medical centers every year, and about over one million of them need to be treated in hospitals (Hooton, 2003; Kunin, 1994; Patton et al., 1991). In addition, women are susceptible to the urinary tract infections because their urinary tract has the characteristic structure of being short and wide and is thus easily infected with bacteria. For this reason, women have a 4- to 10-fold higher incidence of the urinary tract infections than men. Over 50% of adult females visit hospitals due to diseases caused by the urinary tract infections, and the majority of the urinary tract diseases is related with recently increased sexual behavior and contraceptive use of menstruating women (Hooton et al., 1996; Kunin, 1994; Stamm et al., 1993; Uehiling et al., 1994).
The urinary tract infections are largely classified into the upper urinary tract infections and the lower urinary tract infections. The upper urinary tract infections, such as pyelonephritis, have symptoms including pyrexia, nausea and vomiting, costovertebral angle tenderness, serum antibody increase and WBC casts. Symptoms of the lower urinary tract infections, such as cystitis and urethritis, include dysuria, polyuria, increased urinary urgency, and suprapubic discomfort (Hooton, 2003).
In addition, according to the infection states, the urinary tract infections are divided into two subcategories: uncomplicated forms (acute uncomplicated cystitis and acute uncomplicated pyelonephritis) and chronic complicated forms (Stamm et al., 1993). The uncomplicated urinary tract infections occur frequently in people in their twenties and thirties suffering from the urinary tract infections. In contrast, the chronic complicated urinary tract infections are common at all ages due to their underlying primary diseases including urolithiasis, hydronephrosis, bladder tumor, vesicoureteral reflux, neurogenic bladder and prostatic hypertrophy, and, in particular, develop frequently in the elderly or men (Gupta et al., 1999b; Haley et al., 1985). The acute uncomplicated cystitis has symptoms including systemic fever, painful urination, increased urinary frequency, haematuria and pyuria by inflammation, and becomes better by proper antibiotic administration (Stamm et al., 1993). However, the acute uncomplicated nephritis has symptoms including high fever, flank pain and bladder irritation, and often exhibits recurrent microbial reinfection after treatment, resulting in transition to chronic infection. About over 60% of the acute uncomplicated nephritis is easily recurrent, and thus, develops to chronic complicated urinary infections accompanied by fever, frequent pyuria and bacteriuria leading to deterioration of renal diseases, and causes focal segmental glomerulosclerosis (FSG) accompanied by proteinuria and necrosis (Kunin, 1994). Further, about 50% of uropathogenic E. coli are resistant to kanamycin, and 10% of patients with pyelonephritis and cystitis are reinfected within two to three years. In addition, about 10% of these patients suffer from the diseases all their life due to reinfection (Haley et al., 1985; Hooton et al., 1996).
Treatment of the acute uncomplicated urinary tract infections is carried out with the aim of killing pathogens and reducing reinfection. In particular, the treatment aiming to prevent reinfection has been reported to be very important in treatment of the urinary tract infections. Typically, when not treated for a certain period, the urinary tract infections rapidly recur, and this recurrence is believed to be caused by novel E. Coli or bacteria strains. The treatment mainly by antibiotic administration results in disappearance of bacteriuria within 24 hrs, whereas pyuria or other associated symptoms last for several days (Hooton, 2003).
In case of pyelonephritis as another acute uncomplicated urinary tract infection, infections occur in deep regions of the kidney and the urinary epithelium, and, in this case, parenteral treatment is carried out for several days. This parenteral treatment typically takes two weeks or longer. Chemotherapy with trimethoprimsulfamethoxazole (TMP/SMX) (Bactrim®) is more effective than treatment with antibiotics such as ampicillin (Gupta, et al., 1999a, 1999b; Hooton, 2003; Nicolle, 2003). In addition, aminoglycosides, cephalosporins and quinolone are used in therapy of pyelonephritis (Nicolle, 2003).
In case of the chronic complicated infections, treatment is performed by using general antibiotics or chemotherapy, but is highly dangerous because drug administration for a long period increases development of side effects and complications (Kunin, 1994). These treatments have the following problems: (1) emergence of antibiotic-resistant bacteria; (2) an increase in treatment cost by frequent reinfection; and (3) high infection rate (0.5%–0.7% every year). In this regard, there is an increasing need for the development of vaccines effective in treating the urinary tract infections (Hooton et al., 1996; Kunin, 1994; Patton et al., 1991).
To date, there is no commercialized vaccine against uropathogenic E. coli, and only candidate vaccines are at the preclinical stage. Vaccines against uropathogenic E. coli should be prepared by the following development strategy: first, it is preferable that an adhesin essential for bacterial survival is used as a protein antigen; second, a protein antigen should be highly immunogenic and non-toxic; third, a protein antigen should induce mucosal immune responses against a microorganism inhabiting at the junctions between mucosal epithelial cells; fourth, since single use of a protein antigen mostly results in insufficient immune responses, the antigen should be used in combination with an adjuvant capable of enhancing immunogenicity; and, fifth, a protein antigen should be prepared as an oral vaccine convenient upon administration and having no side effects (Service, 1997).
On the other hand, uropathogenic E. coli produces Gal—Gal pili, which participates in its specific attachment to the epithelium of the upper urinary tract, and hemolysin, which is involved in disruption of various cells and intracellular invasion (Roberts et al., 1994). Recently in Korea, using these proteins, vaccine development was attempted by genetic recombination and peptide synthesis, but the vaccine was found to have low antigenicity. In foreign countries, Lagermann et al. (2000) have studied to develop a vaccine using the FimH protein of uropathogenic E. coli by genetic recombination (Kunin, 1994; Patton et al., 1991). The research group recently reported the vaccination effect of FimH against the urinary tract infections in cynomolgus monkeys (Kunin, 1994). According to this report, when MF59 as an adjuvant and FimH were administered to four experimental animals, high vaccination effect was found in three of them. However, in this case, the FimH protein antigen is inconvenient because it should be administered along with the adjuvant to achieve the effective vaccination. In particular, for oral administration, a protein antigen should induce mucosal immune responses and be used essentially along with a nontoxic adjuvant capable of enhancing immunogenicity of a co-administered protein antigen (Foss et al., 1999).
Immune response-associated cells constitute a tissue or organ system to perform effectively their functions, which is called “lymphoid system”. The lymphoid system is classified into the primary (or central) lymphoid system (the thymus and the bone marrow), which substantially produces and differentiates lymphocytes, and the secondary (or peripheral) lymphoid system (the spleen, lymph nodes, mucosal lymphoid organs, etc.). The mucosal lymphoid organs amounting to over ⅓ of body lymphoid tissues among the secondary lymphoid system are the place critical for digestion and absorption of a large number of essential nutrients, and function as a physical barrier against harmful impurities and pathogenic microorganisms, and as an immunological barrier important in the body's protective system (Kagnoff et al., 1996). The mucosal lymphoid organs are largely divided into Bronchus-Associated Lymphoid Tissue (BALT) associated with the lung tissue and alveolar cells in the airways, Nasal-Associated Lymphoid Tissue (NALT) localized at the region where the palate is connected to the nose, and Gut-Associated Lymphoid Tissue (GALT) (Kiyono et al., 1996). On the other hand, Bienenstock (1984) suggested expressing together BALT and GALT as “Mucosal Associated Lymphoid Tissue (MALT)”. MALT is the largest lymphoid tissue in the body, is present at the mucosal region of the gut, and plays an important role in the protection of the body, including triggering IgA immune response in the gut immune system (Mestecky, 1987). On the other hand, among several immune organs in MALT, Peyer's patches is a major lymphoid tissue in the gastrointestinal tract and is an inductive site for sIgA production, and the GI lumen dome is covered with the flattened epithelium containing M cells specialized for antigen absorption (de Haan et al., 1995; Frey et al., 1997; Roit et al., 1992). The M cells facilitate lymphoid cell activation by transporting captured soluble antigens, bacteria or viruses from the lumen to lymphocytes (Kerneis et al., 1997). That is, lymphocytes in Peyer's patches in the gut are activated by reaction with the antigens ingested by the M cells and then differentiated and maturated in the germinal center of lymphatic follicles. The Peyer's patch lymphocytes move rapidly from the mucosal membrane and activate precursor sIgA+ B cells and CD4+ Th cells, move to the mesenteric lymph node (MLN), and enter the thoracic duct (TD) to arrive in the blood stream and circulate through the body (Kerneis et al., 1997). The circulating cells enter an IgA effector site and transport sIgA. Eventually, the gut immune system including Peyer's patches protects the gut, and regulates systemic inflammation and thus effectively inhibits allergic response, autoimmune diseases, and the like.
Most of vaccine proteins against microorganisms inhabiting musocal surfaces are degraded by GALT, or are not absorbed (de Haan et al., 2000; Kerneis et al., 1997; Kunin, 1994). However, cholera toxin (CTX) produced by Vibrio cholera and heat-labile enterotoxin (LTX) produced by E. coli, which are known to be potent adjuvants stimulating mucosal immune responses, induce strong mucosal immune responses by binding to GM1-ganglioside and by tropism of GALT (de Haan et al., 1996; Freytag et al., 1999; Pizza et al., 2001). However, due to toxicity associated with A1 subunit, neither both toxin is used as a mucosal adjuvant in the native form, whereas their variants, A subunit-lacking CTXB and LTXB, or A1 subunit-lacking CTXA2B and LTXA2B are used as adjuvants (Agren et al., 1999; Douce et al., 1999; Haley et al., 1985; Hooton et al., 1996). The CTX A2 and B subunits have been employed in vaccine development. For example, Czerkinsky et al. (1989) genetically replaced the toxic A1 subunit of CTX (CTXA1) by a streptococcal protein adhesin and chemically linked the streptococcal adhesin to CTXB (the nontoxic B subunit of CTX) to provide a vaccine (CTXA2B). In addition, Hajishengallis et al. (1995) and Russell et al. (1991) reported that a genetic recombinant chimeric vaccine, constructed by replacing the CTX A1 subunit (CTXA1) by the saliva-binding region (SBR) of Streptococcus mutans antigen I/II adhesin and linking the SBR to the CTXB by genetic recombination, effectively stimulates the mucosal immune system to secrete secretory IgA antibody (sIgA Ab) via GM1-ganglioside and thus effectively prevents pathogenic bacteria from adhering to mucosal surfaces and forming colonies (de Haan et al., 1995; Harokopakis et al., 1998; Lebens et al., 1994; Saito et al., 2001; Tochikubo et al., 1998; Verweij et al., 1998). According to the research associated with a vaccine against Salmonella typhimurium by Harokopakis et al. (1997), a chimeric protein, constructed by replacing CTXA1 by the SBR of a streptococcal protein AgI/II adhesin and linking the SBR to the CTX A2 and B subunits (CTXA2B) by genetic recombination, strongly stimulates serum IgG and IgA antibody responses in mice. According to the research for developing a vaccine against enterotoxigenic Escherichia coli (ETEC) strains by Hall et al. (2001), after subjects are immunized with a fusion vaccine, ETEC-CTXB, the proportion of vaccinees showing IgA seroconversion ranged from 70 to 96% in children and from 31 to 69% in adults, while IgG seroconversion was observed in 44 to 75% of the vaccinated children and in 25 to 81% of the vaccinated adults. In addition, an animal test with a chimeric protein formed by genetically linking the hpa adhesin of Helicobacter pylori to CTXA2B, conducted by Kim et al. (2001), resulted in an increase in both serum IgA antibody levels as well as sIgA antibody levels in the gastromucosal membrane, demonstrating that the adhesin-CTXA2B chimeric protein is a potential vaccine against H. pylori. Further, Lee et al. (2003) reported that a chimeric vaccine constructed by genetically coupling the S1 fragment of pertussis toxin to CTXA2B induces effective vaccination in mice.
In addition, the LTX A2 and B subunits have been employed in vaccine development. For example, Loregian et al. (1999) constructed a chimeric protein (LTXB-Pol) consisting of the LTX B subunit fused to a 27-mer peptide (antiviral peptide) derived from the DNA polymerase of herpes simplex virus 1 (HSV-1) by genetic recombination. Viral DNA synthesis takes place in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The LTXB-Pol chimeric protein retained the functional properties of both LTXB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. These results indicate that LTXB can be used as a protein carrier and show a potential for HSV vaccine development.
The CTXA2B and LTXA2B subunits have the following advantages as adjuvants: (1) they are able to increase permeability of mucosal epithelial cells (Lycke, 1997); (2) they induce antigen presentation by stimulating MHC class II expression and increasing IL-1 production (Bromander et al., 1991; Millar et al., 2001; Nashar et al., 1993); and (3) they are able to induce mucosal immune responses by stimulating B cells to produce sIgA antibody (Haley et al., 1985; Langermann, 1996; Roit et al., 1992). Therefore, these properties of the adjuvants are applicable to the development of vaccines against the urinary tract infections (Hess et al., 2000).
Many harmful factors are involved in the development and progress of the urinary tract infections caused by E. coli, but, first of all, proper colonization of uropathogenic E. coli must occur (Kunin, 1994; Patton et al., 1991). The E. coli colonization is initiated by attachment to mucosal surfaces via attachment of a specific adhesin of to a specific receptor, mannose, expressed on mucosal surfaces of a host. This colonization of the uropathogenic E. coli is limited in infection sites and tissues (Abraham et al., 1985; Beachey et al., 1981; Beachey et al., 1988; Wizemann et al., 1999). On the other hand, the adhesin of E. coli is usually composed of proteins in the form of fimbriae or fibrillae, and the receptor to which the specific adhesin of E. coli attaches is composed of a glycolipid or glycoprotein (Beachey et al., 1988; Thankavel et al., 1997).
The specific adhesin of the uropathogenic E. coli interact in a lock-and-key fashion with a complementary receptor on mucosal surfaces of the host or tissues thereof for colonization (Abraham et al., 1988; Beachy et al., 1988; Jones et al., 1993; Jones et al., 1995; Kunin, 1994). Two classic examples of bacterial adherence to the epithelial cell surfaces are the lipoteichoic acid (LTA)-mediated attachments of Streptococci and the type 1 fimbriae-mediated attachment of E. coli. In streptococci, the adhesin, LTA, interacts through its lipid moiety with fibronectin molecules bound to the epithelial cells. In type 1 fimbriated E. coli, a minor 29-kDa protein, FimH adhesin, located at the tip of the fimbriae, interacts with D-mannose residues of glycoprotein receptors on host cells (Beachey et al., 1988; Krogfelt et al., 1990). The specific adhesin proteins shown in uropathogenic E. coli and other bacteria are known to be highly conserved (Abraham et al., 1988; Kunin, 1994; Palaszynski et al., 1998).
The research using a FimH knockout variant by Langermann et al. (1997) revealed that the surface protein of uropathogenic E. coli, FimH, plays a central role in its attachment to the urinary tract and colonization. In a test of this research, when the FimH protein was inhibited, bacterial infection was reduced by over 90%. Many other reports demonstrated that the adhesin proteins on bacteria play important roles in the early phase of their infection process (Jones-Carson et al., 1999; Minion et al., 1986; Palaszynski et al., 1998). In particular, the FimH adhesin, located at the tip of type 1 pili of uropathogenic E. coli and other bacteria, specifically interacts with D-mannose residues of glycoprotein receptors on the bladder epithelial tissue, and this interaction is directly involved in bacterial urinary tract infections (Langermann et al., 2001; Knudsen et al., 1998). In particular, the FimH complexed with FimC was suggested as a vaccine candidate capable of preventing the bacterial urinary tract infections. This is because FimC, which serves as a periplasmic chaperone, is critical for proper folding and stabilization of the full-length adhesin (Langermann et al., 2001).
Currently known genes encoding antigenic determinants as virulence factors of uropathogenic E. coli include the FimH adhesin of the type 1 pili, which interacts with D-mannose residues of glycoprotein on the mucosal epithelial cells of host cells, PapG adhesin of P-fimbriae (Hultgren et al., 1989), heat-labile toxin (LTX), heat-stable toxin (STX), aerobactin, haemolysin, serum resistance and KI capsule (Abraham et al., 1988; Hutgren et al., 1993).